The long-term goal of our research is to understand the molecular mechanisms through which G-protein coupled receptors (GPCRs) are activated and attenuated. These receptors represent the largest family in the human genome, and they are the target of most pharmaceutical drugs. We focus our studies primarily on the GPCR rhodopsin and its affiliate proteins. Although crystal structures of key proteins involved in visual signaling are now known, most of the critical structural changes these proteins undergo during their activation and attenuation remain largely a matter of speculation. In particular, we lack even rudimentary information about the dynamic events involved in attenuating rhodopsin signaling, namely, the mechanisms through which retinal is released from the opsin-binding pocket, and how retinal binding and release affects arrestin binding and release. Understanding these processes is of fundamental importance for vision research - the stability of the retinal linkage varies widely among different opsins and is a factor in some visual disease states. Furthermore, although much is known about the mechanism and kinetics of arrestin binding to rhodopsin, little is known about what makes arrestin release after binding, and how this release is related to the status of the retinal chromophore. In Aim I of this proposal we will determine how rhodopsin controls the hydrolysis of its retinal Schiff base linkage. In Aim II we will examine how retinal uptake and release occurs in rhodopsin, using the recent structure of opsin to guide our studies. Finally, in Aim III, we will use our novel methods to follow up on a discovery we made during the last funding period - that arrestin can bind to MIII rhodopsin, thus trapping and preventing retinal release. Understanding how arrestin regulates retinal release is fundamentally important to health, as arrestin may serve to limit the release of free retinal under bright light conditions, and thus help limit the formation of oxidative retinal adducts that can contribute to diseases like atrophic age-related macular degeneration (AMD). Similarly, understanding what makes arrestin "let go" after binding rhodopsin is also crucial - stable rhodopsin-arrestin complexes have been suggested to be a contributing factor in apoptosis and autosomal dominant retinitis pigmentosa (ADRP). PUBLIC HEALTH RELEVANCE: The proposed research will define the molecular events involved in the binding and release of retinal. We will investigate how retinal gets into and out of the binding pocket in rhodopsin, what makes it stay there (i.e., how the Schiff-base linkage forms and hydrolyzes), and how the protein arrestin affects these processes. Answering these fundamental questions will help shed light on more general questions in vision, such as why rates of retinal binding and release vary so greatly between Rod and Cone rhodopsins, and help elucidate underlying mechanisms for several retinal diseases.